Red lettuce

ABSTRACT

The present invention relates to a head-forming lettuce plant of the species  Lactuca sativa  having red leaves throughout the head, including the heart. The red leaves in the heart are red even in the absence of radiation with wavelengths shorter than 400 nm, wherein the absence of radiation with wavelengths shorter than 400 nm is in the growing environment during the complete period from sowing until observation. The ratio between anthocyanin and chlorophyll is between 4 and 50, preferably between 9 and 27. The invention also relates to progeny of the plant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/052,690 filed Mar. 20, 2008 which is a continuation-in-part ofInternational application no. PCT/EP2006/009217, filed Sep. 20, 2006,published as WO 2007/039137 on Apr. 12, 2007, and claiming priority toEP 05077135.1, filed Sep. 20, 2005.

All of the foregoing applications, as well as all documents cited in theforegoing applications (“application documents”) and all documents citedor referenced in the application documents are incorporated herein byreference. Also, all documents cited in this application (“herein-citeddocuments”) and all documents cited or referenced in herein-citeddocuments are incorporated herein by reference. In addition, anymanufacturer's instructions or catalogues for any products cited ormentioned in each of the application documents or herein-cited documentsare incorporated by reference. Documents incorporated by reference intothis text or any teachings therein can be used in the practice of thisinvention. Documents incorporated by reference into this text are notadmitted to be prior art.

FIELD OF THE INVENTION

The present invention relates to a red lettuce plant, to the head oflettuce that can be obtained therefrom and to the gene complex that isresponsible for the red colour of the lettuce.

BACKGROUND OF THE INVENTION

Lifestyles change and the demand from restaurants and catering firms forcolourful and interesting garnish and even from the housewife forready-to-use processed salads continue to rise. As a result, thebreeding companies are looking for varieties with prominent colour,better taste and a wide variety of texture. The lettuce market can bedivided into three groups, namely entire whole heads, pre-cut wholeheads and baby leaves.

At present the attractive red colour in pre-cut lettuce mixtures isoften provided by the presently available “red” lettuce (Lactucasativa), or by radicchio rosso (Cichorium intybus), red cabbage or redchard. The “red” lettuce that has been known so far is not really redthroughout the leaves. In particular the head forming types are eitheronly red along the leaf edges or speckled with red. They are nevercompletely red in the heart. This is caused by the fact that the redcolour is only expressed in those parts of the head that are exposed todaylight, more specifically to UV-radiation. Leaves of the so-called“red” lettuces are therefore for the main part green and their red doeshardly contribute to the red colour impression of a pre-cut lettucemixture.

Often the transitional stage between the red part and the green part ofthe leaf is brown-coloured. This brown colour is considered as visuallyunattractive. Red speckled lettuces are associated with plant diseasesor bloodstains. At present plant breeders select against the browncolour as well as against the speckled red colouring. In addition, it iseven found that speckled or brown coloured lettuce leaves are oftendiscarded, both in the lettuce packaging industry and by the consumer.

The disadvantage of using leaves from other vegetables than lettuce isthat the different taste of these other vegetables is often experiencedas undesirable. Radicchio rosso is for example a Cichorium inlybus thathas a bitter taste. The texture of red cabbage is entirely differentfrom the much softer texture of lettuce.

Anthocyanin synthesis in lettuce is induced by UV-radiation (see forexample Voipio & Autio, 1995, Responses of red-leaved lettuce to lightintensity, UV-A radiation and root zone temperature. In: ActaHorticulturae 399. Greenhouse environmental control and automation. Eds.B J Bailey, T Takakura. Kyoto, Japan. p 183-187; Benoit, et al., 1998,Effect of a photoselective greenhouse film on a few vegetable crops inthe Belgian North Sea climate. In: 14th International congress onplastics in agriculture, Tel Aviv, Israel, March 1997. Laser PagesPublishing, Jerusalem, Israel. p 81-92; Krizek et al., 1998, Inhibitoryeffects of ambient levels of solar UV-A and UV-B radiation on growth ofcv. New Red Fire lettuce. Physiologia Plantarum 103(1), p 1-7; Kleinhenzet al., 2003, Variety, shading, and growth stage effects on pigmentconcentrations in lettuce grown under contrasting temperature regimens.Hortechnology 13(4), p 677-683.). The development of a red colour inmany plant species is dependent on the production of anthocyanin.

The many different varieties of lettuce are often grouped into threetypes. The most common is head lettuce of which there are the crisp head(or iceberg) and butter head. Romaine lettuce (or cos lettuce) forms aloose upright head. The “leaf” lettuce types are non-heading and looseleafed.

In head-forming lettuce types the heart of the head is to a more orlesser extent closed and cannot be reached by light. It is thus notpossible to have anthocyanin production in closed heads such as iceberglettuce and butter lettuce or in romaine lettuce, which has a looselyclosed head.

A related problem is the lack of red colouration of red lettuce grown inglasshouses, plastic tunnels, or in closed containers. Due to the lackof UV-radiation, which is reflected by the glass or plastic covering orwhich is lacking in the artificial assimilation light spectrum, theexpression of anthocyanin is much less than in outdoor conditions. Thisreduces the possibilities to produce red lettuce under indoorconditions, and therefore the possibility to produce red lettuceprotected from adverse weather conditions, like cold, heat, and allsorts of precipitation.

SUMMARY OF THE INVENTION

The present invention now provides a red lettuce, having red leavesthroughout the head, including the heart. The heart leaves of redlettuce of the invention are in essence completely red, which means thatthey contain a uniquely high anthocyanin/chlorophyll-ratio. This is verysurprising because light, more specifically UV-light, which is deemednecessary for the synthesis of anthocyanin is not able to penetrate intothe heart of the head. This demonstrates that in the lettuce of theinvention a light-independent mechanism is responsible for theproduction of anthocyanin.

This light-independency also allows for the cultivation of red lettuce,headed or non-headed, under glass or plastic or in closed containers.The latter uses artificial assimilation lighting which usually givesproblems with the development of red colour. This is due to the factthat the UV-wavelengths (280-400 nm) which are normally essential forinduction of anthocyanin synthesis, are more or less lacking in thelight spectrum.

Under glass or plastic a similar lack of UV-radiation is commonly causedby strong UV-interception of the covering. However, due to itsUV-independent anthocyanin expression the red lettuce of the inventionis suitable for glasshouse, plastic house, and closed containercultivation. This is due to the light-independent mechanism of thepresent invention, which will also be indicated as “UV-independentanthocyanin expression”, or “UV-independent red colouration”.

Deposits

The Deposits with DEPOSITORY, under deposit accession number NCIMB41337, NCIMB 41338 and NCIMB 41339 were made pursuant to the terms ofthe Budapest Treaty. Upon issuance of a patent, all restrictions uponthe deposit will be removed, and the deposit is intended to meet therequirements of 37 CFR §§1.801-1.809. The deposit will be maintained inthe depository for a period of 30 years, or 5 years after the lastrequest, or for the effective life of the patent, whichever is longer,and will be replaced if necessary during that period.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further illustrated in the non-limitingexamples that follow and that refer to the following figures:

FIG. 1 is a graph that shows the distribution of within-line segregationof red colouration of outer and inner leaves for population of 212F3-lines obtained from cross ‘99P.30637×cv. ‘Sharp Shooter’.

FIG. 2 is a diagram that shows the genetic model for anthocyaninexpression based on segregation in cross ‘99P.30637’×cv. ‘SharpShooter’.

FIGS. 3A-3F show representative examples of young plants from the lines03P82421, Pierrot, Pippo, Gringo, Apache and Bijou, respectively.

FIG. 4 shows a comparison of representative examples of a young leaffrom a plant of the present invention with young leaves from varietiesPippo, Pierrot and Gringo.

FIGS. 5A-5D show a comparison of representative examples of a plant ofthe invention (01P80164) and the variety Darkland (99R10044).

FIG. 6A-6D show a comparison of representative examples of a plant ofthe invention (01P80164) and the variety Pierrot (95G2237).

FIG. 7A-7D show a comparison of representative examples of a plant ofthe invention (01P80164) and the variety Roxy (99R10283).

FIG. 8A-8D show a comparison of representative examples of a plant ofthe invention (01P80164) and the variety Sierra (95G1986).

DETAILED DESCRIPTION

In a particular embodiment of the invention, the absorbance ratioA523/A665 between anthocyanin and chlorophyll is between 4 and 50,preferably between 9 and 27.

Whether the expression of anthocyanin in the present invention iscompletely independent of UV-radiation, or that it is triggered by muchlower levels of UV-radiation than normally required for anthocyaninexpression in regular red lettuce plants, is not completely known yet.However, for practical usefulness of the invention this difference doesnot matter.

Although the invention is particularly useful in head-forming lettucetypes, the light-independent red colouring of the invention can also beused in other lettuce types such as leaf lettuce and in baby-leafproduction, which is the production of young lettuce plants forharvesting small, immature leaves.

The pedigree of a red lettuce of the invention is given in Example 1.

The invention relates to all progeny of the original parents that havered leaves in the heart of the head and that meet one or more of theother requirements of the invention, such as the ratio betweenchlorophyll and anthocyanin of the heart leaves.

In addition, non-heading, loose leaf or baby lettuce plants that havethe UV independent red colouration of the invention are also consideredprogeny of the completely red head-forming lettuce plants and thus partof this invention.

The selection of lettuce plants that already are or can lead to thelettuce plants of the invention can be based on visual selection ofred-coloured young plants (step 1). Growing them under glass or plasticcan subsequently distinguish between UV-dependent anthocyaninexpression, i.e. poor red colouration, and UV-independent anthocyaninexpression, i.e. a relatively strong red colouration. These latterplants are planted and grown until they are headed. Then visualselection of plants with red heart leaves is performed by cutting thetop off each plant. The red-hearted plants are then selected to produceoffspring seed.

Crosses are chosen to increase the level of red colouration, and/or toobtain the required level of heading of the mature plant.

As used herein, a lettuce plant is defined as headed, when it has got an‘open head’ or a ‘closed head’, according to the guidelines of UPOV forthe characteristic ‘head formation’ ('TG/13/9. Lettuce (Lactuca sativaL.)). Guidelines for the conduct of tests for distinctness, uniformityand stability.’ International Union for the Protection of New Varietiesof Plants. Geneva. 2004).

The lettuce hearts of the present invention have an absorbance ratioA523/A665 between chlorophyll and anthocyanin which is preferably higherthan 9, more preferably 13 or higher. In normal red lettuce this ratiowas never found to be higher than 3. The chlorophyll and anthocyaninconcentrations used in determining the ratio of the invention aredetermined by means of spectrophotometry. Sample preparation andanalysis is presented in Example 2.

It is to be noted that anthocyanin levels as high as in the heart leavesof the present invention can possibly be found in other lettucevarieties, which are non-heading. Some examples of such non-headingvarieties are 40-0203103-B (Knerr L D, 2005. Lettuce cultivar40-0203103-B. US Patent application US 2005/0144672 A1), Galactic, NewRed Fire, Rolina (Kleinhenz et al, 2003. Variety, shading and growthstage effects on pigment concentrations in lettuce grown undercontrasting temperature regimens. Horttechnology 13(4) p. 677-683), RedSalad Bowl, and Sesam (Voipio I. and Autio J., 1995. Responses ofred-leaved lettuce to light intensity, UV-A radiation and root zonetemperature. Acta horticulturae 399. p. 183-187).

However, these varieties have heart leaves with very high chlorophylllevels, in comparison with the heart leaves of the present invention, asa result of high light exposure due to the non-heading characteristic.On the other hand, heading varieties can possibly be found withsimilarly low chlorophyll levels in the heart leaves as in the heartleaves of the present invention. However, the heart leaves of theseheading varieties do not show the high anthocyanin levels in comparisonwith the heart leaves of the present invention. Due to the low lightexposure of the heart leaves anthocyanin synthesis is normally poorlyinduced. In the present invention, the anthocyanin production isUV-independent. This explains why the anthocyanin/chlorophyll-ratio ofthe present invention is on an unprecedentedly high level.

This new form of red colouration that is UV-independent is found anddeveloped in headed lettuce plants and differs from the red colourationas presented in the prior art. However, the invention also relates tonon-headed lettuce plants, such as loose leaf or baby leaf lettuceplants or lettuce plants that are grown in glass houses or under plasticwhere the amount of UV is lower that have the feature “UV-independentred colouration” of the invention.

Red colouration of the heart leaves was also scored on the RHS colourchart (The Royal Horticultural Society, London, UK). Heart leaf laminacolour, especially on the top of leaf, of the present invention wasscored as 183A, 184A, or 187B, all in the greyed-purple group. Thecolour of the rest of the leaf lamina was scored as 180B, 180C, 180D, or181C, all in the greyed-red group.

It is also possible to score the red colouration and anthocyaninexpression on plants grown under glass or plastic, i.e. conditions withreduced levels of UV-radiation, when compared to the light in outdoorconditions. Also here the plants of the present invention can showhigher levels of anthocyanin and a darker red colour than regular redlettuce. Especially newly appearing leaves of the plants of the presentinvention are much more red than the newly appearing leaves of commonred lettuce plants.

A genetic analysis of the red lettuce of the invention was performed asdescribed in Example 2. It was found therein that at least three genesare involved in the red colouring of the heart of the head.

The present invention thus relates to lettuce plants that are capable ofexpressing anthocyanin and have in addition at least the three genesthat are involved in the red colouring of the heart of the head.Preferably, a plant of the invention comprises the complete gene complexdescribed in Example 2.

It is possible to assess the presence of the loci involved in redcoloration of the present invention by closely linked DNA-markers, likeAFLP, RFLP, RAPD, SCAR, CAPS, SSR, or SNP. For instance, a marker-traitlinkage analysis in the population mentioned above, and its offspringcan provide such closely linked DNA-markers.

The presence in a lettuce plant of a similar genetic constitution of redcolouration of the inner leaves as in the present invention, i.e.assessing whether a lettuce plant is a plant according to the inventioncan easily be assessed by comparison of the phenotype of such potentialplant of the invention with the phenotype of a known plant of thepresent invention. The phenotype can be assessed by, for example, thered colouration of the inner leaves, the anthocyanin/chlorophyll-ratioof the heart leaves, and/or the red colouration of plants grown underglass or plastic.

Another way of assessing the similarity of the genetic constitutionbetween a potential plant of the invention and a known plant of theinvention is by comparison of the marker genotype of the potential plantwith the marker genotype of a known plant of the present invention. Themarker genotype is defined by a set of DNA-based markers, like AFLP,RFLP, RAPD, SCAR, CAPS, SSR, or SNP, which are closely linked to theloci which involved in the red expression of the present invention.

Another way of assessing the similarity of the genetic constitution, isthe comparison of the genotype of a potential plant of the inventionwith the genotype of a known plant of the present invention. Thisgenotype comparison is done on an F2-population, derived byself-fertilising an F1-plant from a cross between the potential plantand a known plant of the present invention. The F2-population can beinvestigated for absence of segregation for the phenotype, i.e. the redcolouration of the inner leaves. In all comparisons phenotypes can alsobe assessed by, for example, the anthocyanin/chlorophyll-ratio of theheart leaves, or the red colouration in plants grown under glass orplastic.

Seed of red lettuce plants according to the invention was deposited withthe NCIMB on 18 Jul. 2005 under accession numbers NCIMB 41337, NCIMB41338 and NCIMB 41339.

The invention also relates to progeny of these seeds, as well as toplants that have obtained the genetic constitution or gene complex ofplants of the invention that leads to the red colouring according to theinvention, either by crossing or by means of molecular biologicaltechniques. The invention also relates to the progeny of these plantsthat have maintained or acquired the trait of red colouring of theinvention.

To transfer the genes that are responsible for the red colour to anotherplant backcross breeding can be used. For this a desirable homozygouscultivar or inbred is the recurrent parent. The source of the trait tobe transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parent(red colouring of the leaves in the heart) are selected and repeatedlycrossed (backcrossed) to the recurrent parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. In case theinheritance of the red colouration is more complex than the inheritanceof the other desired trait, or combination of traits, the parent withred-coloured heart leaves can be used as recurrent parent, and theparent with the other desired trait, or combination of traits, can beused as donor parent. Descriptions of other breeding methods that arecommonly used for different traits and crops can be found in one ofseveral reference books (e.g., “Principles of Plant Breeding” John Wileyand Son, pp. 115-161, 1960; Allard, 1960; Simmonds, 1979; Sneep et al.,1979; Fehr, 1987).

In greater detail, FIG. 1 shows distribution of within-line segregationof red colouration of outer and inner leaves for population of 212F3-lines obtained from cross ‘99P.30637’×cv. ‘Sharp Shooter’. Threeclassifications of red colouration were applied on the population: ‘redoutside’, i.e. within-line frequency of plants with red outer leaves,irrespective of inner leaf colour; ‘red outs&red ins’, i.e. within-linefrequency of plants with red outer and red inner leaves, ‘red ins/redouts’, i.e. frequency of plants with red inner leaves within theline-total of plants with red outer leaves. O=observed; E=expected,according to genetic model.

FIG. 2 shows the genetic model for anthocyanin expression based onsegregation in cross ‘99P.30637’×cv. ‘Sharp Shooter’.

FIG. 3 a shows a plant of the invention. This young plant is a plantfrom a seed from the line 03P82421, which was obtained after threegenerations of self fertilisation of plant 99P38154. FIGS. 3 b, 3 c, and3 d show young plants of parents Pierrot, Pippo, and Gringo,respectively. FIGS. 3 e and 3 f show young plants of comparisonvarieties Apache and Bijou, respectively. All these young plants weregrown on peat blocks in a growth chamber with 14 h light at 16° C. and10 h dark at 12° C. Light was produced by Philips TLD 36 W 840 REFLEXtubes, with 1 tube per 0.24 square m, at 0.6 m distance above theplants.

FIG. 4 shows a young leaf of a plant of the invention, indicated by‘NEW’, in comparison to young leaves from the commercial varietiesPippo, Pierrot and Gringo. These young plants were grown on peat blocksin a growth chamber with 14 h light at 16° C. and 10 h dark at 12° C.Light was produced by Philips TLD 36 W 840 REFLEX tubes, with 1 tube per0.24 square m, at 0.6 m distance above the plants.

FIGS. 5-8 show comparisons between a plant of the invention (01P80146)and the commercial varieties Darkland (indicated by ‘99R10044’; FIGS. 5a-5 d), Pierrot (indicated by ‘95G2237’; FIGS. 6 a-6 d), Roxy (indicatedby ‘99R10283’; FIGS. 7 a-7 d) and Sierra (indicated by ‘95G1986’; FIGS.8 a-8 d), respectively. The a-figures (5 a, 6 a, 7 a, 8 a) are showingthe full harvested heads, The b-figures (5 b, 6 b, 7 b, 8 b) are showingthe heart without outer leaves, The c-figures (5 c, 6 c, 7 c, 8 c) areshowing a longitudinal section of the heart. The d-figures (5 d, 6 d, 7d, 8 d) are showing cut heart leaves. The lettuce plants of theinvention are completely red in the heart of the head whereas the otherlettuce plants are not. All plants are grown in Aramon, France in 2002:sown on 10 Jan. 2002, transplanted into a confidential open field on 15Feb. 2002, harvested on 25 Apr. 2002.

EXAMPLES Example 1 Pedigree of a Red Lettuce of the Invention

The lettuce of the invention was obtained according to the followingpedigree: in 1986 a cross was made between a plant of cv. Pippo (RijkZwaan; red-coloured) and a plant of cv. Blonde Maraichere (Caillard;green-coloured).

In 1988 a red offspring plant from this cross was used as a father in across with a plant of cv. Gringo (Rijk Zwaan; red-coloured). In the sameyear a cross was made between a plant of cv. Pierrot (Rijk Zwaan;red-coloured) and a plant of cv. Roxette (Rijk Zwaan; green-coloured).

In 1989 a red offspring plant from this cross was used as a father in across with a plant of cv. Krizet (Rijk Zwaan; green-coloured).

In 1992 a red F3-offspring plant was selected from theKrizet×(Pierrot×Roxette)-cross mentioned above and it was used as amother in a cross with a father plant, Which was a selected redoffspring plant from the Gringo×(Pippo×Blonde Maraichere)-crossmentioned above. A red F4-plant from this newly obtained 1992-cross wasselected in 1995 and used as a mother in a cross with a plant of cv.Roxette (Rijk Zwaan; green-coloured).

Selection on type, heading, and colour, which was performed in the F2-,F3-, and F4-generation from this cross, resulted in a red F4-plant(98P.31582) in 1998. The F5-line appeared to be segregating for colour(red vs. green), but selection in the next generation resulted in threeheaded F5-plants with completely red heart leaves (99P.38152; 99P.38154;99P.30637), which were multiplied in the years afterwards.

The offspring showed no segregating green plants, and seeds weredeposited under nrs. 02R.2413, 01R.1439, 02R.2418. These F5-plants andtheir offspring have served as a parent source for further breeding oflettuce with light-independent red colouration.

In conclusion, it can be said that none of the used parent varieties,i.e. Pippo, Blonde Maraichere, Gringo, Pierrot, Roxette, and Krizet,have the characteristic red leaves in the heart of the head. Theinvention comprises the unique and new combination of genes from thesered and green parent varieties, which is providing the completely redleaves in the heart of the head.

Example 2 Genetic Analysis of the Red Lettuce of the Invention

As used herein, a locus (plural: loci) is defined as the specific placeon a chromosome where a gene is located. (Griffiths A J F, Miller J H,Suzuki, D T, Lewontin R C, Gelbart, W M. ‘An introduction to geneticanalysis.’ 6^(th) edition. 1996. WH Freeman and Company, New York.) Thenumber of loci responsible for red colouration in a given plant can beestablished by a genetic analysis of the offspring of a cross of thisplant with the green-coloured cv ‘Sharp Shooter’.

A genetic analysis of the red lettuce of the invention was performed asfollows: a cross was made between an offspring plant from F5-plant nr.99P.30637, i.e. a plant of the present invention, and a non-brilliantgreen-coloured iceberg lettuce plant of cv. Sharp Shooter (S V S,Waycott et. al., 1999: U.S. Pat. No. 5,973,232). From the offspring ofthis cross 212 random F2-plants were multiplied into F3-lines.

These 212 F3-lines were evaluated in a confidential outdoor trial inFijnaart, the Netherlands. Seeds were sown on peat blocks on Jun. 17,2002, young plants were raised in a glasshouse, and transplanted in thefield on Jul. 5, 2002. The harvest date (time of observation) was onAug. 19 until Aug. 23, 2002. The plot size was 24 plants per F3-line,i.e. 4 rows of 6 plants. Lines were not replicated, because expressionof colour traits is very stable within one trial. Observations were doneper F3-plant.

Observed traits were: a) colour, and, if red, its intensity andexpression pattern on the outer leaves, b) colour, and, if red, itsintensity and expression pattern on the inner leaves, c) degree ofheading, d) in case of green outer leaves: scoring in 2 classes:brilliant or non-brilliant.

The intensity and expression pattern of the red colouration was scored,in increasing order, as: 1) tinged or blushed, i.e. light redcolouration on the outer and inner leaf edges, or on the inner leafbase, 2) red spotted, 3) green spotted, i.e. green spots on red leafsurface, 4) fully red, i.e. intensely red coloured without spots, on thelight-exposed part of the outer leaf parts, and, in case of innerleaves, throughout the inner leaf.

Heading was scored between 1) slightly open heading, like Romaine or coslettuce, and 2) strong heading with clearly overlapping leaves, likeiceberg lettuce.

Some plants in the trial died before harvest. Of the intended 5088plants, i.e. 212×24, in total 5007 F3-plants reached harvestable stageand were scored (see Table 1).

All plants showed heading levels, which were at least comparable toRomaine or cos lettuce. Data showed a segregation of 55 fully greenlines out of the total of 212. Out of the 157 lines that were having atleast one plant with red colouration, 28 lines comprised of plants whichwere all showing anthocyanin expression on the outer leaves. Out ofthese 28 lines, 8 lines comprised of plants which were all showinganthocyanin expression on the heart leaves. The red-coloured plants outof 33 of the 157 lines with at least one red-coloured plant, wereshowing anthocyanin expression only in the outer leaves and not in theheart leaves. The red-coloured plants out of another 33 of the 157 lineswere always showing anthocyanin expression on both the outer leaves aswell as the heart leaves. The red-coloured plants out of the remaining91 lines all showed anthocyanin expression in the outer leaves. However,these plants showed a within-line segregation of anthocyanin expressionin the heart leaves. Not a single plant out of the 5007 plants showedanthocyanin expression in the heart leaves in combination withgreen-coloured outer leaves, which had no anthocyanin expression.

It is therefore concluded that one or more genes that result inanthocyanin expression in the outer leaves, are required for anthocyaninexpression in the heart leaves. In the case of segregation between redand green, segregation ratios between red and green ranged from 23:1 to1:22 for colouration of outer leaves. For inner leaves the segregationratios between red and green colouration ranged from 18:1 to 1:23,excluding all plants with green outer leaves. Table 1 shows thewithin-line distribution of plants with red and green colouration ofoutside and inside leaves for a population of 212 randomly derivedF3-lines from cross ‘99P.30637’×cv. ‘Sharp Shooter’.

TABLE 1 all plants with part of all plants green outer plants with withred leaves red outer leaves outer leaves all plants with green 55 29 4inner leaves part of plants with red 0 75 16 outer leaves have red innerleaves all plants with red outer 0 25 8 leaves have red inner leaves

These segregation data were used to construct a genetic model, under theassumption of independent Mendelian segregation ratios. It was foundthat at least three loci are involved in obtaining UV-independentanthocyanin expression in the inner leaves. The first locus, furtherindicated as A-a, is also found in known red lettuce and the dominantallele A is required in all cases to obtain expression of anthocyanin.Probably this locus is the C or G-locus (Robinson et al., 1983, Thegenes of lettuce and closely related species. In: Plant BreedingReviews 1. Ed. J. Janick. p 267-293).

A second locus is found by fitting the model, further indicated as B-b,where the homozygous presence of recessive allele b results inanthocyanin expression of outer leaves in combination with A. Two moreloci, further indicated as C-c and D-d, were found to be also involvedin anthocyanin expression in the outer leaves. Either presence of atleast one copy of the dominant allele C, or homozygous presence of therecessive allele d results in anthocyanin expression in the outerleaves, but only in the case that also at least one copy of allele A ispresent.

Three more loci, further indicated as E-e, F-f, and G-g, were found. Toobtain UV-independent anthocyanin expression in the inner leaves thepresence of at least one A-allele, and two b-alleles is required, incombination with either the presence of one copy of the dominantE-allele, or the homozygous presence of the f-allele, or the homozygouspresence of the g-allele. So the presence of alleles for red colourationon at least three loci, namely A-a, B-b, and either E-e, F-f, or G-g, isrequired for UV-independent anthocyanin expression in the inner leaves.Furthermore, it is assumed that at least three, but probably all sevenloci mentioned above are involved in the intensity of red colouring ofthe present invention.

Example 3 Determination of Colour, Anthocyanins and Chlorophylls inLettuce

1. Sample Preparation and Analyses

A spectrophotometer UltrospecIII (Pharmacia), with the followingspecifications was used:

-   -   Monochromator: Czerny Turner with holographic diffraction        grating (1200 lines/mm)    -   Wavelength accuracy: ±1 nm    -   Wavelength reproducibility: ±0.5 nm    -   Detector type: single solid state silicon photodiode    -   Bandwidth: 5 nm

2. Principle

The red colour (anthocyanins) and green colour (chlorophylls) aredetermined with a biochemical method. Two extracts are made, one formeasuring the absorbance at 523 nm which is a measure for totalanthocyanins and one for measuring the absorbance at 665 nm which is ameasure for total chlorophylls (chlorophyll a and b).

3. Sample Preparation

The headed and mature lettuce plant is harvested and outer leaves aretaken off, until the heart is left over. The heart of the lettuce isused for the analysis. The heart leaves should not have been exposed todirect sunlight before harvest, except for a small tip of the leaf(maximum 10%) on the top of the plant. A heart contains at least 10leaves with a length of 1 cm or longer. The oldest leaves of the heartshould be concave.

The hearts are put in plastic bags and frozen at <−70° C. After at leasta few days in <−70EC the frozen hearts are pulverized with asledgehammer. The fine sample is grinded in a Grindomix (GM 200, Retsch,5″ 3000 rpm followed by 5″ 5000 rpm) using the free floating lid inpresence of liquid nitrogen (sample should be kept frozen) to obtain apowder.

The powder is then placed in a tube and the tube with powder is chilledin liquid nitrogen and optionally stored at <−70° C. until analysis.

4. Analysis

Three grams of the powder are weighed into four tubes of 50 ml. Twotubes are used for the analysis of anthocyanins and two tubes for theanalysis of chlorophylls.

For measurements of anthocyanins 1.0 M HCl in 50% methanol isimmediately added to the two tubes. 5-10 ml/g sample is used dependenton the colour of the sample and extract. The used volume (ml) is noted.The sample solution is mixed by hand and put on ice. A part of thesolution is put in a 1.5 ml eppendorf tube and the tube is centrifugedat 4° C., 13000 rpm for 4 min.

Spectra are measured with a spectrophotometer with a band width of 5 nm.A 1 cm cuvette is used.

The spectrum of 360-900 nm is measured and the absorbance at 523 nm (ifnecessary after dilution with extraction liquid) and the maximumwavelength (8_(max)) are determined. 8_(max) should be close to 523 nm.

Chlorophylls are measured by immediately adding 100% methanol to the twotubes. 5-10 ml/g sample methanol is used dependent on the colour of thesample and extract. The volume (ml) used is noted. The sample solutionis mixed by hand and sonificated in an ultrasonic bath for 5 min at ‘setdegas’. A part of the solution is put in a 1.5 ml eppendorf tube andcentrifuged at 4° C., 13000 rpm for 4 min.

The spectrum of 360-900 nm is measured and the absorbance at 665 nm (ifnecessary after dilution with extraction liquid) and the maximumwavelength (8_(max)) determined. 8_(max) should be close to 665 nm.

The A523 and A665 are corrected to represent the absorbance of asolution of 1 g fresh weight in 10 ml extraction volume. The correctedA523 and A665 are calculated with a correction for weighted sample (ing), extraction volume (in ml) and if necessary dilution using thefollowing formula:

Correction:

$A_{{corrected} \cdot} = {\frac{A_{measured}*{{extr}.{volume}}}{{weight}*10}*{dilution}}$

The ratio A523/A665 is calculated.

Table 2a shows the chlorophyll and anthocyanin absorbance andanthocyanin/chlorophyll-ratio observed on lettuce in Aramon, France(sowing 10 Jan. 2002, transplanting 15 Feb. 2002, harvested 25 Apr.2002). Line 01P.80146 is an offspring line obtained from plant 99P.38154by 2 generations of self-fertilisation. Pierrot (Rijk Zwaan), Darkland(Central Valley), Sierra (Vilmorin), and Roxy (Enza) are common lettucevarieties.

Table 2b shows chlorophyll and anthocyanin absorbance andanthocyanin/chlorophyll-ratio observed on lettuce grown in Fijnaart, theNetherlands (sowing 21 May 2002, transplanting 10 Jun. 2002, harvested20 Aug. 2002). Line 01P.80146 is an offspring line obtained from plant99P.38154 by 2 generations of self-fertilisation. Pierrot (Rijk Zwaan),Pippo (Rijk Zwaan), Red Rosalita (Johnny's Selected & Orsetti), Darkland(Central Valley), Sierra (Vilmorin), and Roxy (Enza) are common lettucevarieties.

TABLE 2a id. nr. (breeding) 01P.80146 Pierrot Darkland Sierra Roxy Totalchlorophyll A665 Mean 0.105 0.396 0.198 0.476 0.228 Stdev 0.028 0.0730.062 0.038 0.080 Min 0.083 0.321 0.132 0.451 0.155 Max 0.150 0.4860.279 0.520 0.353 Nr 5 5 4 3 5 Total anthocyanin A523 Mean 1.589 0.8830.006 0.033 0.087 Stdev 0.195 0.123 0.001 0.006 0.030 Min 1.370 0.7250.005 0.029 0.063 Max 1.890 1.070 0.006 0.041 0.137 Nr 5 5 4 3 5 Totalanthocyanin/ Total chlorophyll: A523/A665 Mean 16.16 2.27 0.03 0.07 0.38Stdev 4.88 0.38 0.01 0.01 0.01 Min 9.13 1.82 0.02 0.06 0.36 Max 20.172.68 0.04 0.08 0.40 Nr 5 5 4 3 5

TABLE 2b id. nr. (breeding) Sierra Roxy Darkland 01P.80146 Red RosalitaPippo Pierrot Total chlorophyll A665 Mean 0.570 0.267 0.343 0.063 0.2780.456 0.325 Stdev 0.189 0.105 0.101 0.013 0.075 0.244 0.113 Min 0.3450.180 0.205 0.049 0.205 0.244 0.196 Max 0.836 0.405 0.447 0.082 0.3880.823 0.505 Nr 5 5 5 5 5 5 5 Total anthocyanin A523 Mean 0.038 0.0460.010 1.228 0.030 0.693 0.870 Stdev 0.019 0.020 0.004 0.145 0.014 0.4370.277 Min 0.028 0.027 0.005 1.042 0.017 0.371 0.474 Max 0.072 0.0800.013 1.422 0.052 1.422 1.231 Nr 5 5 5 5 5 5 5 Total anthocyanin/ Totalchlorophyll: A523/A665 Mean 0.07 0.18 0.03 20.08 0.12 1.53 2.69 Stdev0.02 0.05 0.01 4.09 0.08 0.46 0.31 Min 0.05 0.11 0.02 15.41 0.06 1.042.42 Max 0.09 0.22 0.05 26.18 0.25 2.23 3.10 Nr 5 5 5 5 5 5 5

Deposit Information

The F5-plants 01R.1439, 02R.2413 and 02R.2418 were deposited on 18 Jul.2005 with the NCIMB, Ferguson Building, Craibstone Estate, Bucksburn,Aberdeen AB21 9YA, United Kingdom under the deposit accession numbersNCIMB 41337, NCIMB 41338 and NCIMB 41339, respectively.

Various modifications and variations of the described products andmethods of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in chemistry, biology orrelated fields are intended to be within the scope of the followingclaims.

1. A head-forming lettuce plant of the species Lactuca sativa having redleaves throughout the head, including the heart.
 2. The lettuce plant ofclaim 1 having red leaves in the heart, even in the absence of radiationwith wavelengths shorter than 400 nm.
 3. The lettuce plant as claimed inclaim 2, wherein the absence of radiation with wavelengths shorter than400 nm is in the growing environment during the complete period fromsowing until observation.
 4. The lettuce plant as claimed in claim 1,wherein the absorbance ratio A523/A665 between anthocyanin andchlorophyll of the heart leaves is between 4 and
 50. 5. The lettuceplant as claimed in claim 4, wherein the absorbance ratio A523/A665between anthocyanin and chlorophyll of the heart leaves is between 9 and27.
 6. The lettuce plant as claimed in claim 1, wherein the absorbanceratio A523/A665 between anthocyanin and chlorophyll of the ten youngestheart leaves larger than 1 cm is between 4 and
 50. 7. The lettuce plantas claimed in claim 6, wherein the absorbance ratio A523/A665 betweenanthocyanin and chlorophyll of the ten youngest heart leaves larger than1 cm is between 9 and
 27. 8. The lettuce plant as claimed in any claim 1having a genetic constitution that leads to red colouring of the leavesin the heart and which comprises a gene locus for anthocyaninexpression, at least two gene loci that are involved in the redcolouring in the heart of the head and optionally at least two gene locithat are involved in the intensity of red colouring.
 9. The lettuceplant as claimed in claim 1, obtainable from seeds as deposited on Jul.18, 2005 with the NCIMB under accession numbers NCIMB 41337, NCIMB 41338and NCIMB
 41339. 10. The lettuce plant as claimed in claim 1 having thegenetic constitution of the seeds as deposited on Jul. 18, 2005 with theNCIMB under accession numbers NCIMB 41337, NCIMB 41338 and NCIMB 41339that leads to red colouring of the leaves in the heart of the head. 11.The lettuce plant of the species Lactuca sativa having red leaves ofclaim 1, even in the absence of radiation with wavelengths shorter than400 nm in the growing environment during the complete period from sowinguntil observation.
 12. The lettuce plant as claimed in claim 11, whereinthe absorbance ratio A523/A665 between anthocyanin and chlorophyll isbetween 4 and
 50. 13. The lettuce plant as claimed in claim 11, whereinthe absorbance ratio A523/A665 between anthocyanin and chlorophyll isbetween 9 and
 27. 14. The lettuce plant as claimed claim 11, wherein theabsorbance ratio A523/A665 between anthocyanin and chlorophyll of theten youngest heart leaves larger than 1 cm is between 4 and
 50. 15. Thelettuce plant as claimed in claim 14, wherein the absorbance ratioA523/A665 between anthocyanin and chlorophyll of the ten youngest heartleaves larger than 1 cm is between 9 and
 27. 16. Progeny of lettuceplants as claimed in claim
 1. 17. Seeds of the lettuce plants as claimedin claim
 1. 18. A head of the lettuce plants as claimed in claim
 1. 19.A gene complex which in a lettuce plant of the species Lactuca sativaleads to red coloration of the leaves throughout the head, including theheart which comprises a gene locus for anthocyanin expression, at leasttwo gene loci that are involved in the red colouring in the heart of thehead and optionally at least two gene loci that are involved in theintensity of red colouring.
 20. The gene complex as claimed in claim 19as present in the seeds as deposited on Jul. 18, 2005 with the NCIMBunder accession numbers NCIMB 41337, NCIMB 41338 and NCIMB 41339.